The chemical modification of proteins, nucleotides, and other biomolecules is an important research tool in immunology, histochemistry, biochemistry, and cell biology. Conjugation is a form of modification in which two or more molecules with distinct properties are joined so that some of the characteristics of each joined molecule are retained in the product. For example, molecules with special properties of fluorescence or binding specificity can be covalently bound or conjugated to a protein, DNA strand, or other biomolecule. Proteins, peptides, polysaccharides, hormones, nucleic acids and their analogs, and liposomes may be conjugated with each other or with molecular groups that add useful properties (e.g., drugs, radionuclides, toxins, fluorphores, photoprobes, inhibitors, enzymes, haptens, ligands).
Fluorescent dyes are routinely conjugated to biological molecules to impart the properties of light absorption and fluorescence to the biomolecule for a variety of purposes including separation and fluorescence imaging. For example, fluorescent labeling of antibody molecules is used extensively for applications such as immunohistological staining and cell separation by flow cytometry.
The chemical modification of biomolecules with fluorescent probes generally involve covalent attachment of the probe to the biomolecule, although many fluorophore probes for nucleic acids do not covalently bond to the target biomolecule. The most commonly exploited conjugation points for covalent bonding in proteins are the aliphatic amine groups, notably, the .epsilon.-amine groups of lysine and the .alpha.-amino groups of the N-terminal amino acids. Other conjugation points in proteins include thiol residues in cysteine and cystine, the phenolic hydroxyl group of tyrosine and carboxlic acid groups of aspartic acid and glutamic acid.
Fluorescent probes are chosen based upon their physical properties. The probe must be able to bind to a particular site on a biomolecule (i.e., amine or thiol group of a protein) and must be relatively specific for that particular binding site. A probe that is too reactive in the conjugation reaction solution (e.g. an aqueous solution) will hydrolyze before binding to the target site. In most cases, the fluorescent probe should also be relatively soluble in water so as not to precipitate out of solution and also be capable of reacting rapidly and specifically with a particular binding site at substantially neutral pH (and particularly physiological pHs).
The reaction kinetics of a fluorescent probe should be controllable. In other words, the amount of fluorescent label conjugated per biomolecule should be capable of being regulated. Regulation of the reaction kinetics is important to permit consistent reproducibility of labeling results. Regulation is also important because excess labeling can alter the biological characteristics of the biomolecule, e.g., it may lower the biomolecule's affinity for its target. In the case of labeling antibodies, for example, excessive substitution of the fluorescent probe to the antibody can reduce the affinity of the antibody for a particular antigen.
The fluorescent probe should have a relatively large extinction coefficient, that characterizes its light-absorbing power, at least in certain uses (e.g. fluorescent flow cytometry). The quantum yield should be large when the probe is bound to the target and is in the solvent environment where the fluorescence measurement is to be made. The fluorescent probe should also excite at desired wavelength levels, preferably above 500 nanometers to avoid autofluorescence from cell constituents. The photostability, or the number of excitations that a fluorescent probe can withstand is also important, especially for detecting a small number of probes in solution.
Common fluorescent labels include fluoroscein and rhodamines. Fluorescein has a relatively high extinction coefficient and quantum yield, and is generally soluble in aqueous solutions and easily conjugated to proteins. However, fluorescein is relatively photounstable and loses fluorescence below pH 8. Unfortunately, it is preferred to use a pH below 8 when a probe is conjugated to a protein in a living cell, and consequently fluoroscein's use in this context has some disadvantages. Fluorescein also has a wavelength of excitation in a region that produces autofluorescence.
Rhodamines excite in the 500 to 600 nanometer range, where less autofluorescence is generated. Rhodamines are more photostable than fluorescein and are pH insensitive under physiological conditions (pH 7-8). However, rhodamines have a relatively low quantum yield and limited solubility.
One of the most widely used rhodamines for amine-reactive fluorescent labeling is a sulfonyl halide derivative of rhodamine, sulforhodamine 101 acid chloride, or Texas Red (a trademark of Molecular Probes, Inc.). Sulforhodamine 101 compounds like Texas Red are particularly useful as fluorescent dyes since their spectra minimally overlap the spectra of fluorescein and other green dyes. Typically, sulforhodamine101 compounds exhibit fluorscence in the wavelength range of 610-630 nanometer. In comparison, fluorescein has a wavelength of exitation in the range of 500-520 nanometers. Therefore, fluorescein and sulforhodamine 101 are well-suited for two-color imaging and cell sorting.
Texas Red forms stable sulfonamide bonds that make it a popular amine-modifying reagent and thus it has proven to be very useful to label lysine groups on proteins and polypeptides generally. However, sulfonyl halides, like Texas Red, are highly reactive, are less specific, and hydrolyze easily prior to conjugation to the target site. Texas Red not only selectively reacts with amines, but also, through a competing reaction, tends to react with the solvent medium.
Further, the labeling of proteins with Texas Red is not kinetically controllable. The labeling results cannot be consistently reproduced and it is impossible to maintain the substitution ratio of Texas Red to biomolecule in any desired range depending on input ratios. Instead, the substitution ratio of Texas Red on a protein depends on intangibles like stir rate, order of mixing, and the size of the reaction particles.
Thus, there is a need for a rhodamine fluorescent probe that is kinetically controllable, is site specific but only moderately reactive, and will not hydrolyze too rapidly. ##STR2##